PhiC31 integrase-mediated transgenesis systems

PhiC31 integrase-mediated transgenesis systems are based on the site-specific bacteriophage PhiC31 integrase which mediates sequence-directed, irreversible and highly efficient integration between a bacterial attachment site (attB) and a phage attachment site (attP). Injecting plasmid containing attB site and white marker into attP-containing docking site strain(s) with PhiC31 activity makes the resultant stable w+ transformants containing your gene-of-interest between attL and attR sites (irreversible).

We use M{vas-int.Dm}ZH-2A as the genomic PhiC31 source if the attP site stock does not contain it. If the screening marker is fluorescence-related, we may use the genomic P{nos-phiC31\int.NLS}X source (non-fluorescence), please let us know if this is the case.

For vermillion+ markers, e.g. for creating stable attP-gRNA lines, you may actually choose any of the vermillion+ based attP lines from these below lists. We may setup the crosses to the y-v- line and screen for v+. You may get the same v+ G2 transformants as using the v- based attP lines. This is just regular Plan H or Plan I with extra v+ screening. You may also ask us to keep the resulting gRNA lines and have us cross to any of the Cas9 stocks and inject their F1s (additional Plan R) later.

P{CaryP}
Two attP landing sites were created by the Calos group. These two sites were initially created by the traditional P-element system. See below paper for more info.

PhiC31-mediated RMCE
In June, 2006, the Wu lab published a method that targets constructs to predetermined genomic sites using the phiC31 integrase system in conjunction with Recombinase Mediated Cassette Exchange (RMCE).

P[acman]
P[acman] system makes use of the combined tools of a conditional amplifiable technique for typical P1 and BAC construct, recombineering, and PhiC31-mediated trangenesis. It has the advantage of delivering large DNA fragments into specific genomic locations with attP site, which were generated by the Ballen lab and markered by yellow. Here we are incorporating the P[acman] system with the work from the Basler group which used endogenous PhiC31 source instead of co-injecting the PhiC31 mRNA.

MiMIC
MiMIC is a highly versatile transposon insertion resource for engineering Drosophila melanogaster genes. MiMIC system was developed by the collaboration among the laboratories of Hugo Bellen (Baylor College of Medicine), Roger Hoskins (Lawrence Berkeley National Laboratory) and Allan Spradling (Carnegie Institution of Washington).

FlyC31
In FlyC31 system, a library of landing platforms containing attP site throughout the Drosophila genome were created by the Basler group, which are ready for the transgenesis of the construct with attB site. These landing platforms were deliberately designed to be manipulated in vivo by Cre/loxP system after transgenesis to get rid of most of the non-necessary sequences and only keep your gene-of-interest flanking with one loxP and one attL site.

The PhiC31 system and the Gateway System
One of the "Gateway-PhiC31 system", for example, can be found here. Please note the attP1, attP2, attB1, attB2, etc, at the original Murphys Gateway are of different concepts.

#FlyBase Drosophila melanogaster (R6.10)1Eye color were determined three days after eclosion (heterozgous state)6Transformation efficiency Score was calculated based on actual running orders and will be updated quarterly. Average score and >30kb BACs scores were shown

Currently available Wu strains and Bateman strains (PhiC31-RMCE strains) at BestGene Inc.

#FlyBase Drosophila melanogaster (R6.10)7Eye color were determined three days after eclosion. This is the color of the original stock, not the transformant6Transformation efficiency Score was calculated based on actual running orders and will be updated quarterly. Average score and >30kb BACs scores were shown

Currently available Bellen strains (P[acman] strains) at BestGene Inc.

#FlyBase Drosophila melanogaster (R6.10)1Eye color were determined three days after eclosion (heterozgous state)2These lines were very hard to transform6Transformation efficiency Score was calculated based on actual running orders and will be updated quarterly. Average score and >30kb BACs scores were shown12Donated by Jack Bateman 10/9/2009. Recombined single sites of 9724

#FlyBase Drosophila melanogaster (R6.10)3According to FlyC31 website4These strains are weak; BDSC#24486 is the weakest strain (low survival rate after injection)5Enhancer trap effect from a nearby gene hibris and is not useful for reporter gene analysis and other applications where ectopic expression is an issue6Transformation efficiency Score was calculated based on actual running orders and will be updated quarterly. Average score and >30kb BACs scores were shown11M{vas-int.Dm}ZH-2A removed and loxP-3xP3-RFP-loxP removed version12M{vas-int.Dm}ZH-102D removed and loxP-3xP3-RFP removed version

Currently available Perrimon strains (P{CaryP} strains) at BestGene Inc.

#FlyBase Drosophila melanogaster (R6.10)1Eye color were determined three days after eclosion (heterozgous state)6Transformation efficiency Score was calculated based on actual running orders and will be updated quarterly. Average score and >30kb BACs scores were shown

*Some lines contain the nos-PhiC31 genomic integrase source as indicated at the Genotype.#FlyBase Drosophila melanogaster (R6.10)1Eye color were determined three days after eclosion (heterozgous state)6Transformation efficiency Score was calculated based on actual running orders and will be updated quarterly. Average score and >30kb BACs scores were shown9This strain has very high non-specific integration rate. Service W (PCR confirmation of integration site) is highly recommended10Service W is NOT available for this site